A Propidium Monoazide (PMAxx)-Droplet Digital PCR (ddPCR) for the Detection of Viable Burkholderia cepacia Complex in Nuclease-Free Water and Antiseptics
Abstract
:1. Introduction
2. Materials and Methods
2.1. ddPCR Assay
2.2. Optimization of PMAxx Treatment
2.3. Limit of detection (LOD) of PMAxx-ddPCR
2.4. Assessment of the PMAxx-ddPCR Assay
2.4.1. Using Various Ratios of Live vs. Dead B. cenocepacia J2315 Cells
2.4.2. Using Different Concentrations of CHX and BZK
2.5. Application of the PMAxx ddPCR Assay with 20 BCC Strains in Nuclease-Free Water and Antiseptics
2.6. Statistical Analysis
3. Results
3.1. Evaluating the Primer Sets
3.2. Optimization of the PMAxx Concentration and Light Exposure Time
3.3. LOD of PMAxx-ddPCR
3.4. Assessment of the PMAxx-ddPCR Assay
3.4.1. Assessing Live/Dead Cells in Nuclease-Free Water
3.4.2. PMAxx-ddPCR Assay in CHX and BZK
3.5. Application of PMAxx-ddPCR Assay in Nuclease-Free Water and Antiseptics
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Amplicons Name | Primer Sequence (5—3) | Amplicon Size (bp) | |
---|---|---|---|
RibB67 | Forward Reverse | GCGATACGAAGGAACACCTG CGTAGCCGGACATGCTG | 189 |
RibB5 | Forward Reverse | GGCCGGATGGTGATCCT GTCATCAGCGGCAGGTG | 176 |
Tested Inoculum (CFU/mL) | Nuclease-Free Distilled Water | CHX | BZK | |||
---|---|---|---|---|---|---|
Without PMAxx | With PMAxx | Without PMAxx | With PMAxx | Without PMAxx | With PMAxx | |
104 | 50/60 a | 38/60 | 54/60 | 42/60 | 48/60 | 50/60 |
103 | 55/60 | 34/60 | 49/60 | 23/60 | 47/60 | 54/60 |
102 | 49/60 | 37/60 | 41/60 | 11/60 | 28/60 | 33/60 |
10 | 50/60 | 35/60 | 35/60 | 16/60 | 29/60 | 24/60 |
204/240 (85.0%) b | 144/240 (60.0%) | 179/240 (74.6%) | 92/240 (38.3%) | 152/240 (63.3%) | 161/240 (67.1%) | |
p = 0.0163 c | p = 0.0001 | p = 0.7166 |
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Daddy Gaoh, S.; Kweon, O.; Lee, Y.-J.; Hussong, D.; Marasa, B.; Ahn, Y. A Propidium Monoazide (PMAxx)-Droplet Digital PCR (ddPCR) for the Detection of Viable Burkholderia cepacia Complex in Nuclease-Free Water and Antiseptics. Microorganisms 2022, 10, 943. https://doi.org/10.3390/microorganisms10050943
Daddy Gaoh S, Kweon O, Lee Y-J, Hussong D, Marasa B, Ahn Y. A Propidium Monoazide (PMAxx)-Droplet Digital PCR (ddPCR) for the Detection of Viable Burkholderia cepacia Complex in Nuclease-Free Water and Antiseptics. Microorganisms. 2022; 10(5):943. https://doi.org/10.3390/microorganisms10050943
Chicago/Turabian StyleDaddy Gaoh, Soumana, Ohgew Kweon, Yong-Jin Lee, David Hussong, Bernard Marasa, and Youngbeom Ahn. 2022. "A Propidium Monoazide (PMAxx)-Droplet Digital PCR (ddPCR) for the Detection of Viable Burkholderia cepacia Complex in Nuclease-Free Water and Antiseptics" Microorganisms 10, no. 5: 943. https://doi.org/10.3390/microorganisms10050943
APA StyleDaddy Gaoh, S., Kweon, O., Lee, Y. -J., Hussong, D., Marasa, B., & Ahn, Y. (2022). A Propidium Monoazide (PMAxx)-Droplet Digital PCR (ddPCR) for the Detection of Viable Burkholderia cepacia Complex in Nuclease-Free Water and Antiseptics. Microorganisms, 10(5), 943. https://doi.org/10.3390/microorganisms10050943